The long term goals of this research are to identify the common sites and mechanisms of chemical interference with reproductive endocrine function in vertebrates, using two teleosts, Atlantic croaker and spotted seatrout (Cynoscion nebulosus), as models. The overall aims of this continuation proposal are to determine the extent and nature of xenobiotic interactions with a representative of steroid membrane receptors, the well characterized plasma membrane receptor in spotted seatrout ovaries for the maturation-inducing steroid, 17a, 20B, 21-trihydroxy- 4- pregnen-3-one (20B-S), which regulates final oocyte maturation by a nongenomic mechanism. Specific objectives are to: 1) Test the hypothesis that a broad range of xenobiotics can interfere with 20B-S action by binding to the 20B-S membrane receptor. 2) Investigate the significance of localization of the 20B-S receptor in the plasma membrane on its interactions with lipophilic xenobiotics and steroids. 3) Compare the binding characteristics of the partially purified 20B-S membrane receptor to those of classic nuclear steroid receptors. 4) Test the hypothesis that estrogenic xenobiotics bind to other classes of steroid receptors in addition to the estrogen receptor to disrupt reproductive processes. 5) Determine the effects of several xenobiotics which bind to the 20B-W receptor on 20B-S-induced final oocyte maturation, ovulation and reproductive success in vivo. The binding of estrogenic xenobiotics, nonestrogenic xenobiotics with varying degrees of lipophilicity, antiestrogens and C21 steroids with various substitutions to the progestin membrane receptor will be examined in competition assays and competitive inhibitors identified. Xenobiotic agonists and antagonists of 20B-S action will be identified after short term exposure (1-5 minutes) in modified in vitro final oocyte maturation bioassay which can distinguish receptor-mediated from nonspecific toxic actions of xenobiotics. In addition, the reproductive consequences of 20B-S receptor occupancy by xenobiotics will be investigated during hormonal induction of final oocyte maturation and ovulation in vivo. The binding affinities of these compounds for the 20B-S membrane receptor will be compared to their affinities for the solubilized 20B-S receptor and to classical (nuclear) progestin and estrogen receptors and a sex-steroid binding protein in this species. The 20B-S membrane receptor will be partially purified and further characterized to determine its similarity to classic steroid receptors. Protocols for optimal solubilization of the receptor with detergents will be developed and the solubilized receptor will be partially purified by anion exchange, affinity and gel filtration chromatography prior to examination of its ligand specificity and binding characteristics in the receptor assay and estimation of its molecular weight by non-denaturing electrophoresis. These studies will provide the first extensive information on the binding of xenobiotics to an important class of steroid receptors, plasma membrane receptors. Interference with steroid hormone action by xenobiotics at the level of membrane receptors would indicate an additional mechanism by which organochlorines and other xenobiotics can disrupt reproduction and other physiological processes in humans and wildlife.